WO2022063899A1 - Sel pharmaceutique d'un dérivé d'arylpyrrole - Google Patents

Sel pharmaceutique d'un dérivé d'arylpyrrole Download PDF

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WO2022063899A1
WO2022063899A1 PCT/EP2021/076203 EP2021076203W WO2022063899A1 WO 2022063899 A1 WO2022063899 A1 WO 2022063899A1 EP 2021076203 W EP2021076203 W EP 2021076203W WO 2022063899 A1 WO2022063899 A1 WO 2022063899A1
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atropisomer
tartaric acid
composition
acid salt
salt
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PCT/EP2021/076203
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English (en)
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WO2022063899A9 (fr
Inventor
Meriel Ruth Major
Robert George Boyle
Stuart Travers
David Winter Walker
Julian Scott Northen
Stefania SANTONI
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Sentinel Oncology Limited
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Priority to BR112023004275A priority Critical patent/BR112023004275A2/pt
Priority to EP21777561.8A priority patent/EP4217343A1/fr
Priority to KR1020237014101A priority patent/KR20230074784A/ko
Priority to IL301462A priority patent/IL301462A/en
Priority to US18/245,999 priority patent/US20240140908A1/en
Priority to JP2023519234A priority patent/JP2023544291A/ja
Priority to CN202180065652.1A priority patent/CN116348105A/zh
Priority to AU2021350389A priority patent/AU2021350389A1/en
Priority to CA3192729A priority patent/CA3192729A1/fr
Priority to MX2023003300A priority patent/MX2023003300A/es
Publication of WO2022063899A1 publication Critical patent/WO2022063899A1/fr
Publication of WO2022063899A9 publication Critical patent/WO2022063899A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/30—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
    • C07D207/32—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C07D207/33—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms with substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C07D207/337—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
    • C07C59/235—Saturated compounds containing more than one carboxyl group
    • C07C59/245—Saturated compounds containing more than one carboxyl group containing hydroxy or O-metal groups
    • C07C59/255—Tartaric acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/07—Optical isomers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/13—Crystalline forms, e.g. polymorphs

Definitions

  • This invention relates to a pharmaceutical salt of an atropisomer of a tri-aryl pyrrole compound, methods for its preparation, pharmaceutical compositions containing it and its use in treating diseases such as cancer.
  • the protein expressed by the normal KRAS gene performs an essential function in normal tissue signalling.
  • the mutation of a KRAS gene by a single amino acid substitution, and in particular a single nucleotide substitution, is responsible for an activating mutation which is an essential step in the development of many cancers.
  • the mutated protein that results is implicated in various malignancies, including lung adenocarcinoma, mucinous adenoma, ductal carcinoma of the pancreas and colorectal carcinoma.
  • the KRAS protein is a GTPase and is involved in many signal transduction pathways.
  • KRAS acts as a molecular on/off switch. Once it is turned on, it recruits and activates proteins necessary for the propagation of growth factor and other receptors' signals such as c-Raf and PI-3 Kinase. Normal KRAS binds to GTP in the active state and possesses an intrinsic enzymatic activity which cleaves the terminal phosphate of the nucleotide converting it to GDP. Upon conversion of GTP to GDP, KRAS is turned off. The rate of conversion is usually slow but can be sped up dramatically by an accessory protein of the GTPase-activating protein (GAP) class, for example RasGAP.
  • GAP GTPase-activating protein
  • KRAS can bind to proteins of the Guanine Nucleotide Exchange Factor (GEF) class, for example SOS1, which forces the release of bound nucleotide. Subsequently, KRAS binds GTP present in the cytosol and the GEF is released from ras-GTP. In mutant KRAS, its GTPase activity is directly removed, rendering KRAS constitutively in the active state. Mutant KRAS is often characterised by mutations in codons 12, 13, 61 or mixtures thereof.
  • GEF Guanine Nucleotide Exchange Factor
  • PLK1 Polo-Like Kinase 1
  • PLK1 is a serine/threonine kinase consisting of 603 amino acids and having a molecular weight of 66 kDa and is an important regulator of the cell cycle.
  • PLK1 is important to mitosis and is involved in the formation of and the changes in the mitotic spindle and in the activation of CDK/cyclin complexes during the M-phase of the cell cycle.
  • Polo-like kinases contain an N-terminal Serine/Threonine kinase catalytic domain and a C-terminal region that contains one or two Polo-boxes (Lowery et al., Oncogene, (2005), 24, 248-259).
  • the entire C- terminal region, including both Polo-boxes functions as a single modular phosphoserine/threonine-binding domain known as the Polo-box domain (PBD).
  • PBD phosphoserine/threonine-binding domain
  • the PBD inhibits the basal activity of the kinase domain. Phosphorylation-dependent binding of the PBD to its ligands releases the kinase domain, while simultaneously localizing Polo-like kinases to specific subcellular structures.
  • Tumour protein p53 functions as a tumour suppressor and plays a role in apoptosis, genomic stability and inhibition of angiogenesis. It is known that tumours with both p53-deficiency and high PLK1 expression may be particularly sensitive to PLK1 inhibitors (Yim et al., Mutat Res Rev Mutat Res, (2014). 761, SI- 39).
  • the alkylating agent temozolomide (Temodar®, Temodal®) is currently the first line treatment for the brain cancer glioblastoma multiforme and is frequently used in combination with radiation therapy.
  • drug resistance is a major problem in the management of glioblastoma and therefore limits the usefulness of temozolomide. At the present time, therefore, malignant glioblastoma remains incurable.
  • Polo like kinase 1 (PLK1) is overexpressed in a range of tumour types including glioblastoma multiforme (Translational Oncology 2017, 10, 22-32). Furthermore, recent studies have shown that PLK1 drives checkpoint adaptation and resistance to temozolomide in glioblastoma multiforme [Oncotarget 2017, 8, 15827-15837],
  • Ependymomas are tumours of the brain and spinal cord with current standard of care limited to surgery and radiation.
  • PLK1 has been implicated in Ependymomas and inhibitors of PLK1 are active against Ependymoma cell lines [Gilbertson et. al., Cancer Cell (2011) 20, 384-399],
  • PLK1 has also been investigated as a target for Diffuse Intrinsic Pontine Glioma (DIPG), a high grade, aggressive childhood brain tumour [Amani et al. BMC Cancer (2016) 16, 647 and Cancer Biology and Therapy (2016) 19, 12, 1078-1087]
  • DIPG Diffuse Intrinsic Pontine Glioma
  • PLK4 is a polo-like kinase family member of the serine/threonine kinases that plays a critical role in centrosome duplication, acting as a central regulator of centriole duplication (Bettencourt-Dias, Curr Biol. 2005 15(24) ;2199-207). PLK4 dependent alterations in centrosomes can lead to asymmetric chromosome segregation at mitosis, which can trigger cell death after chromosome mis-segregation and mitotic defects.
  • PLK4 is aberrantly expressed in human cancers and is implicated in tumorigenesis and metastasis. As such PLK4 has been highlighted as a promising target for cancer therapy (Zhao, J Cane Res Clin Oncol., 2019).
  • PLK4 is overexpressed in many cancers including rhabdoid tumours, medulloblastoma and other embryonal tumours of the brain (Pediatr Blood Cancer. 2017), as well as breast, lung, melanoma, gastric, colorectal, pancreatic and ovarian cancer. Elevated or hyperactivated PLK4 is associated with poor survival rates in cancer patients, including ovarian, breast and lung cancers (Zhao, J Cane Res Clin Oncol., 2019).
  • PLK4 inhibition has been studied for the treatment of glioblastoma multiforme and it has been demonstrated that PLK4 plays a critical role in the regulation of temozolomide chemosensitivity.
  • the combination of temozolomide with inhibition of PLK4 in glioblastoma PDX models has been shown to enhance the anti-tumor effects compared to temozolomide alone (Cancer Letters, Vol 443, 2019, 91-107).
  • PLK4 is reported to cooperate with p53 inactivation in cancer development, and it is predicted that cancers with PLK4 overexpression and p53 deficiency are prone to form tumours (Serein, 2016; Nat Cell Biol 18:100-110). Therefore, compounds that inhibit PLK4 activity would be anticipated to be useful in the treatment of p53 mutant cancers. Inhibition of PLK4 results in anti-tumour activity in lung cancer, with activity seen in cancers bearing wildtype and mutant KRAS (Kawakami, PNAS 2018, 115(8) 1913- 18). Therefore, compounds that inhibit PLK4 activity would be anticipated to be useful in the treatment of KRAS mutant cancers.
  • ring X is a benzene or pyridine ring
  • ring Y is a benzene, pyridine, thiophene or furan ring
  • Ar 1 is an optionally substituted benzene, pyridine, thiophene or furan ring
  • R 1 to R 4 , R 6 , R 7 are hydrogen or various substituents.
  • the compounds are described as having anti-cancer activity and having good brain exposure after oral dosing, making them good candidates for the treatment of brain cancers.
  • the compounds are active against glioblastoma cell lines and are believed to act as inhibitors of the Polo Box Domain of PLK1 kinase. It is also disclosed that the compounds are active against mutant-RAS cancer cell lines (such as HCT 116) and should also be useful in the treatment of cancers arising from KRAS mutations.
  • Atropisomers can be classified into three categories based on the amount of energy needed for the chiral axis to racemize via rotation and the length of time required for racemization to occur.
  • Class 1 atropisomers possess barriers to rotation around the chiral axis of ⁇ 84 kJ/mol (20 kcal/mol) and racemize over a time period measured in minutes or less at room temperature;
  • Class 2 atropisomers possess a barrier to rotation between 84 and 117 kJ/mol (20-28 kcal/mol) and racemize over a time period measured in hours to months at room temperature;
  • Class 3 atropisomers possess a barrier to rotation >117 kJ/mol (28 kcal/mol) and racemize over a period of time measured in years at room temperature.
  • the nearest substituents either side of the aryl-aryl bond are assigned a priority in the order a-b-c-d.
  • the atropisomer is the S isomer.
  • the substituents a, b and c are in a clockwise arrangement.
  • the atropisomers of 2, 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2- yl]-N-[2 (dimethylamino)ethyl]benzamide and its analogues are sufficiently stable to be isolated and characterised and have been found not to racemize to any significant extent even when heated to temperatures of up to 80 °C for a period of 10 days.
  • the atropisomers can therefore be classified as Class 3 atropisomers.
  • the two individual atropisomers of 2,4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)- phenyl]pyrrol-2-yl]-N-[2 (dimethylamino)ethyl]benzamide have been found to have significantly different biological properties.
  • one of two atropisomers of the pair is significantly more active against certain cancer targets than the other atropisomer of the pair.
  • the atropisomer having better biological activity against the biological targets of interest has been shown by X-ray analysis of a single crystal to have the R configuration, i.e. the chemical structure (1):
  • a (+)-L-tartaric acid salt of 2,4-[5-(4-chlorophenyl)-1- [2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2 (dimethylamino)ethyl]benzamide in which there is an approximately 1 :1 molar ratio between acid and base, has advantages over the free base form of 2,4-[5-(4-chlorophenyl)-1-[2- (trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2 (dimethylamino)ethyl]benzamide and other salts of the compound.
  • the (+)-L-tartaric acid is particularly advantageous in that it is a highly crystalline and stable solid taking up only surface moisture ( ⁇ 1% at 90%RH) with improved water solubility over the free base. These properties render it particularly suitable for pharmaceutical development.
  • the invention provides (/?)- 2,4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N- [2-(dimethylamino)-ethyl]benzamide (+)-L-tartaric acid salt having an approximately 1 :1 molar ratio between acid and base.
  • the invention provides:
  • composition of matter comprising the (+)-L-tartaric acid salt of any one of Embodiments 1.3 to 1.13 wherein either (a) the single atropisomer is the only atropisomer of 2,4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2 (dimethylamino)ethyl]benzamide present in the composition or (b) there is less than 10% by molar amount, relative to the said single atropisomer, of any other atropisomer of 2,4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2 (dimethylamino)ethyl]benzamide.
  • composition of matter according to Embodiment 1.14 wherein either (a) the single atropisomer is the only atropisomer of 2,4-[5-(4-chlorophenyl)-1-[2- (trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2 (dimethylamino)ethyl]benzamide present in the composition or (b) there is less than 5% by molar amount, relative to the said single atropisomer, of any other atropisomer of 2,4-[5-(4-chlorophenyl)-1-[2- (trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2 (dimethylamino)ethyl]benzamide.
  • composition of matter according to Embodiment 1.14 wherein either (a) the single atropisomer is the only atropisomer of 2,4-[5-(4-chlorophenyl)-1-[2- (trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2 (dimethylamino)ethyl]benzamide present in the composition or (b) there is less than 2% by molar amount, relative to the said single atropisomer, of any other atropisomer of 2,4-[5-(4-chlorophenyl)-1-[2- (trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2 (dimethylamino)ethyl]benzamide.
  • composition of matter according to Embodiment 1.14 wherein either (a) the single atropisomer is the only atropisomer of 2,4-[5-(4-chlorophenyl)-1-[2- (trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2 (dimethylamino)ethyl]benzamide present in the composition or (b) there is less than 1.5% by molar amount, relative to the said single atropisomer, of any other atropisomer of 2,4-[5-(4-chlorophenyl)-1-[2- (trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2 (dimethylamino)ethyl]benzamide.
  • composition of matter according to Embodiment 1.14 wherein either (a) the single atropisomer is the only atropisomer of 2,4-[5-(4-chlorophenyl)-1-[2- (trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2 (dimethylamino)ethyl]benzamide present in the composition or (b) there is less than 1% by molar amount, relative to the said single atropisomer, of any other atropisomer of 2,4-[5-(4-chlorophenyl)-1-[2- (trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2 (dimethylamino)ethyl]benzamide.
  • compositions of matter, compounds or salts as defined in any one of Embodiments 1.1 to 1.19 may contain one or more isotopic substitutions, and a reference to a particular element includes within its scope all isotopes of the element.
  • a reference to hydrogen includes within its scope 1 H, 2 H (D), and 3 H (T).
  • references to carbon and oxygen include within their scope respectively 12 C, 13 C and 14 C and 16 O and 18 O.
  • the isotopes may be radioactive or non-radioactive.
  • the composition of matter or atropisomers contain no radioactive isotopes. Such compounds are preferred for therapeutic use.
  • the composition of matter or atropisomer may contain one or more radioisotopes. Compounds containing such radioisotopes may be useful in a diagnostic context.
  • compositions of matter, compounds or salts as defined in any one of Embodiments 1.1 to 1.19 may form solvates and anhydrates.
  • solvates are solvates formed by the incorporation into the solid state structure (e.g. crystal structure) of the compositions of matter or atropisomers of the invention of molecules of a non-toxic pharmaceutically acceptable solvent (referred to below as the solvating solvent).
  • Solvates can be prepared by recrystallising the composition of matter or atropisomers of the invention with a solvent or mixture of solvents containing the solvating solvent. Whether or not a solvate has been formed in any given instance can be determined by subjecting crystals of the composition of matter or atropisomer to analysis using well known and standard techniques such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC) and X-ray powder diffraction (XRPD).
  • TGE thermogravimetric analysis
  • DSC differential scanning calorimetry
  • XRPD X-ray powder diffraction
  • the solvates can be stoichiometric or non-stoichiometric solvates.
  • solvates are those formed with any one of more solvents selected from water, alcohols (e.g. methanol, ethanol and isopropyl alcohol), alkyl-alkanoate esters (e.g. ethyl acetate and isopropyl acetate), ethers (particularly cyclic ethers such as 1-4-dioxane and tetrahydrofuran) and monocyclic lactams (e.g. N- methylpyrrolidone).
  • solvents selected from water, alcohols (e.g. methanol, ethanol and isopropyl alcohol), alkyl-alkanoate esters (e.g. ethyl acetate and isopropyl acetate), ethers (particularly cyclic ethers such as 1-4-dioxane and tetrahydrofuran) and monocyclic lactams (e.g. N- methylpyrrolidone).
  • alcohols e.g. m
  • compositions of matter, compounds or salts as defined in any one of Embodiments 1.1 to 1.19 may be provided in the form of an anhydrate.
  • anhydrate refers to a solid particulate form which does not contain water (and preferably does not contain any other solvents) within its three-dimensional structure (e.g. crystalline form), although particles of the salt or compound may have water molecules attached to an outer surface thereof.
  • the (+)-L-tartaric acid salt of the invention can be prepared from the atropisomer of the formula (1) by reaction with tartaric acid in a solvent or mixture of solvents and then isolating the tartrate salt from the solvent or mixture of solvents.
  • the atropisomer of formula (1) can be dissolved or suspended in one solvent to form a first mixture, and (+)-L-tartaric acid dissolved or suspended in the same or another solvent to form a second mixture, and then the first and second mixtures combined and left (e.g. with stirring) for a period of time to allow salt formation to occur, followed by isolation of the (+)-L-tartaric acid salt.
  • the molar amounts of atropisomer of formula (1) and (+)-L-tartaric acid are approximately equivalent; i.e. there is preferably a 1 :1 molar ratio between the atropisomer of formula (1) and (+)-L-tartaric acid.
  • the (+)-L-tartaric acid salt can be isolated from the combined mixture by filtration (when a precipitate is formed) or by evaporation of the solvents.
  • the different solvents can be selected so as to act as co-solvents or as anti-solvents.
  • the solvent or mixture of solvents can be selected so that they retain the (+)-L- tartaric acid salt at least partially in solution when heated, but then deposit the salt as a precipitate when the solvent or mixture of solvents is cooled.
  • the solvent used to form the first mixture can be selected from, for example, aliphatic ketones, aliphatic esters of aliphatic acids, non-aromatic cyclic ethers and aliphatic alcohols.
  • a particular example of an aliphatic ketone is acetone.
  • Examples of aliphatic esters of aliphatic acids include C2-4 alkyl esters of acetic acid, a particular example being isopropylacetate.
  • non-aromatic cyclic ethers examples include dioxane, 2-methyltetrahydrofuran and tetra hydrofuran, a particular example being 2-methyltetrahydrofuran.
  • aliphatic alcohols are C2-4 aliphatic alcohols, and more particularly C3-4 alkanols such as isopropyl alcohol and butanol.
  • the solvent used to form the second mixture can be selected from, for example, water, non-aromatic cyclic ethers and aliphatic alcohols.
  • a particular example of an aliphatic alcohol solvent for the second mixture is ethanol.
  • a particular example of a non-aromatic cyclic ether solvent for the second mixture is tetrahydrofuran (THF).
  • Another particular example of a solvent for use in forming the second mixture is water.
  • the (+)-L-tartaric acid salt of the atropisomer of formula (1) can exist in several crystalline forms, notably Pattern A (which is a solvate) and Pattern B (which is an anhydrate). Characterising details for the different crystalline forms are provided elsewhere herein.
  • the different crystalline forms can be prepared by varying the solvents and heating conditions used in the formation of the salts.
  • (+)-L-tartaric acid salt of the atropisomer of formula (1) having Pattern A a solution of the atropisomer in acetone is mixed with a solution of (+)-L-tartaric acid in ethanol at a temperature in the range from 20 °C to 30 °C (for example approximately 25 °C), the resulting mixture is stirred or otherwise agitated for a length of time (e.g. 12-24 hours) sufficient to allow salt formation to take place, and the salt is then isolated by filtration.
  • a length of time e.g. 12-24 hours
  • (+)-L-tartaric acid salt of the atropisomer of formula (1) having Pattern A a solution of the atropisomer in isopropyl alcohol is mixed with a solution of (+)-L-tartaric acid in ethanol at a temperature in the range from 35 °C to 45 °C (for example approximately 40 °C), the resulting mixture is cooled to a temperature in the range from 20 °C to 30 °C (for example approximately 25 °C) over a period of approximately 1-3 hours, and the salt is then isolated by filtration.
  • (+)-L-tartaric acid salt of the atropisomer of formula (1) having Pattern A a solution of the atropisomer in 2-methyltetrahydrofuran is mixed with a solution of (+)-L-tartaric acid in ethanol at a temperature in the range from 20 °C to 30 °C (for example approximately 25 °C), the resulting mixture is stirred or otherwise agitated for a length of time (e.g. 12-24 hours) sufficient to allow salt formation to take place, and the salt is then isolated by filtration.
  • a length of time e.g. 12-24 hours
  • (+)-L-tartaric acid salt of the atropisomer of formula (1) having Pattern B a solution of the atropisomer in isopropyl acetate at a temperature in the range from 35 °C to 45 °C (for example approximately 40 °C) is mixed with a solution of (+)-L-tartaric acid in ethanol, the resulting mixture is cooled to a temperature in the range from 20 °C to 30 °C (for example approximately 25 °C) over a period of approximately 1-3 hours, and the salt is then isolated by filtration.
  • a solution of the atropisomer in isopropyl acetate at a temperature in the range from 35 °C to 45 °C (for example approximately 40 °C) is mixed (either portion-wise or in one single charge) with a solution of (+)-L-tartaric acid in THF and one or more seed crystals of the salt Pattern B are added to give a precipitate, the mixture is cooled to a temperature in the range from 20 °C to 30 °C (for example approximately 25 °C) and stirred or agitated for period of time (e.g. 12 to 24 hours, particularly approximately 20 hours) sufficient to allow ripening of the precipitate to a state in which it can be isolated by filtration.
  • period of time e.g. 12 to 24 hours, particularly approximately 20 hours
  • (+)-L-tartaric acid salt of the atropisomer of formula (1) having Pattern B a solution of the atropisomer in butanol at a high temperature in the range from 70 °C to 85 °C (for example approximately 80 °C) is mixed (either portion-wise or in one single charge) with a solution of (+)-L-tartaric acid in water, the resulting mixture is cooled to an intermediate temperature in the range 65° C to 70 °C before adding one or more seed crystals of the Pattern B salt and cooling the mixture to a low temperature in the range from 3-10 °C over a period of 8 to 15 hours, and thereafter stirring or otherwise agitating the resulting mixture at or near the low temperature for a further period of 2 to 8 hours (e.g. approximately 6 hours) and then filtering off the Pattern B salt thus formed.
  • a solution of the atropisomer in butanol at a high temperature in the range from 70 °C to 85 °C (for example approximately 80 °C) is mixed
  • the atropisomer of the formula (1) can be prepared by:
  • Scheme 1 The starting materials for the synthetic route shown in Scheme 1 are 4-cyano- acetophenone (4) and 4-chlorophenacylbromide (5), both of which are commercially available.
  • Step 1 4-cyano-acetophenone (4) and 4-chlorophenacylbromide (5) are reacted together to give 4-[4-(4-chlorophenyl)-4-oxo-butanoyl]benzonitrile (6).
  • the reaction is typically carried out in the presence of a zinc (II) salt (for example, zinc chloride) in a suitable solvent, for example a mixture of a non-polar (e.g. hydrocarbon) solvent such as benzene or toluene and a tertiary alcohol (for example, t-butanol), in the presence of a tertiary amine such as triethylamine.
  • a zinc (II) salt for example, zinc chloride
  • a suitable solvent for example a mixture of a non-polar (e.g. hydrocarbon) solvent such as benzene or toluene and a tertiary alcohol (for example, t-butanol)
  • Step 2 4-[4-(4-chlorophenyl)-4-oxo-butanoyl]benzonitrile (6) is reacted with 2- trifluoromethyl aniline to give 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl) phenyl)- 1 H-pyrrol-2-yl) benzonitrile (7).
  • the reaction is typically carried out in the presence of an acid catalyst such as p-toluenesulphonic acid in a suitable high boiling solvent (for example dioxane) at an elevated temperature (for example between 130 and 170 °C) and/or with microwave irradiation.
  • the reaction may be carried out for between 1 and 12 hours, for example between 1 and 6 hours.
  • Step 3 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl) phenyl)-1 H-pyrrol-2-yl) benzonitrile (7) is subjected to alkaline hydrolysis to give 4-(5-(4-chlorophenyl)-1- (2-(trifluoromethyl) phenyl)-1 H-pyrrol-2-yl) benzoic acid (8).
  • the hydrolysis reaction is typically carried out in an aqueous solvent, which may contain an alcohol such as methanol, in the presence of an alkaline metal hydroxide such as sodium hydroxide (typically in an excess amount), and generally with heating, for example to a temperature in the range from 60-80 °C or a period of up to about 20 hours, or more.
  • an alkaline metal hydroxide such as sodium hydroxide (typically in an excess amount)
  • heating for example to a temperature in the range from 60-80 °C or a period of up to about 20 hours, or more.
  • Step 3 one of two possible routes to the atropisomer (1) can be followed.
  • 4-(5-(4-chlorophenyl)- 1-(2-(trifluoromethyl) phenyl)-1 H-pyrrol-2-yl) benzoic acid (8) is reacted with N,N- dimethylethylenediamine under amide forming conditions to give a racemic mixture of atropisomers of 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl) phenyl)-1 H-pyrrol-2- yl)-N-(2-(dimethylamino) ethyl) benzamide (9) which is then resolved into its individual atropisomers by chiral separation to give the atropisomer (1).
  • racemic 6 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl) phenyl)- 1 H-pyrrol-2-yl) benzoic acid (8) is subjected to a chiral separation to give the atropisomer (3) which is then reacted with N,N-dimethylethylenediamine under amide forming conditions to give atropisomer (1).
  • the carboxylic acids (3) and (8) are reacted with N,N-dimethylethylenediamine under amide forming conditions in the presence of an amide coupling reagent.
  • amide coupling reagents include carbodiimide-based coupling reagents such as 1,3-dicyclohexylcarbo-diimide (DCC) (Sheehan et al, J. Amer. Chem Soc. 1955, 77, 1067) and 1-ethyl-3-(3’-dimethylaminopropyl)-carbodiimide (referred to herein either as EDC or EDCI) (Sheehan et al, J. Org.
  • DCC 1,3-dicyclohexylcarbo-diimide
  • EDC 1-ethyl-3-(3’-dimethylaminopropyl)-carbodiimide
  • the amide coupling reaction is typically carried out in a non-aqueous, polar, non- protic solvent such as tetrahydrofuran or dimethylformamide, or mixtures thereof at room temperature or thereabouts (e.g. 18-30 °C) in the presence of a noninterfering base, for example a tertiary amine such as triethylamine or N,N- diisopropylethylamine.
  • a noninterfering base for example a tertiary amine such as triethylamine or N,N- diisopropylethylamine.
  • Chiral separations of mixtures of atropisomers of carboxylic acid (8) and amide (9) can be carried out using a variety of techniques. For example, chiral chromatography can be used to separate individual atropisomers. The retention times of the atropisomers in the chiral chromatography procedures provide a means of differentiating between and characterising the individual atropisomers whose NMR and MS properties are typically the same.
  • Chiral chromatography columns that can be used to separate the individual atropisomers comprise an immobilised chiral stationary phase (CSF) which can be, for example, based on a functionalised amylose or cellulose.
  • CSF immobilised chiral stationary phase
  • Examples of such CSF’s are amylose and celluloses that have been functionalised with chloro- and/or methyl-substituted phenyl carbamates.
  • Particular examples of chiral columns that may be used to isolate the individual atropisomers of the present invention are the “Chiralpak IG” columns available from Daicel Corporation.
  • Mobile phases that can typically be used in conjunction with the above chiral columns include mixtures of (A) liquid alkanes such as n-heptane containing a small amount (e.g. up 1% (v/v) and more usually about 0.1% (v/v)) of an alkylamine base such as diethylamine; and (B) alcohols and mixtures thereof such as mixtures of isopropyl alcohol and methanol (e.g. 70:30 IPA:MeOH).
  • the mobile phase can comprise a mixture of A:B in the range of ratios 80:20 to 95:5, for example from approximately 85:15 to approximately 90:10.
  • the mobile phases may be used in isocratic or gradient elution methods but, in one embodiment of the invention, are used in an isocratic elution method.
  • the atropisomers of the invention may also be resolved by chiral HPLC under supercritical fluid chromatography (SFC) conditions.
  • the mobile phase comprises a supercritical fluid such as carbon dioxide, often with a co-solvent such as an alcohol or mixture of alcohols, e.g. methanol, ethanol and isopropanol.
  • the Chiralpak IG columns referred to above may be used in SFC chromatography procedures, using carbon dioxide/methanol/isopropanol mixtures as the mobile phase.
  • the Lux family of chiral columns are available from Phenomenex, Inc.
  • YMC Amylose-SA columns are available from YMC America, Inc.
  • chiral chromatography methods may be used in particular for the separation of atropisomer (1) from the racemic mixture (9).
  • a chiral acid e.g. (+)-10- camphorsulphonic acid
  • the racemic mixture (9) can be reacted with the racemic mixture (9) to form a chiral salt as a mixture of diastereoisomers that can be separated by crystallisation to give a salt of atropisomer (1) which can then be converted to the free base of atropisomer (1).
  • the racemic mixture of atropisomers of carboxylic acid (8) can be resolved by forming a salt with a chiral amine such as (S)-1-(4-methoxyphenyl)-ethylamine to form a mixture of diastereoisomers that can be separated by crystallisation, for example with the assistance of seed crystals of the salt of the chiral amine with atropisomer (1).
  • a chiral amine such as (S)-1-(4-methoxyphenyl)-ethylamine
  • a method for the preparation of a compound of the formula (1) which method comprises the reaction of a compound of the formula (3) with N,N- dimethylethylenediamine under amide forming conditions.
  • a method according to Embodiment 2.1 wherein the amide forming conditions include the presence of an amide coupling reagent, for example an amide coupling agent as described herein.
  • a method for the preparation of a compound of the formula (3) which method comprises the chiral separation of the compound of formula (3) from a mixture of atropisomers of formula (8), for example by chiral chromatography or salt formation with a chiral base and resolution of the resulting chiral salt.
  • An atropisomer compound having the formula (3), or a salt thereof for example a metal salt such as an alkaline or alkaline earth metal salt, or a salt with ammonia or an organic amine).
  • the atropisomer of formula (1) and its tartrate salts as defined herein are inhibitors of the polo box domains of PLK1 and PLK4 kinases but do not inhibit the catalytic domains of PLK1 and PLK4 kinases. Since PBD domains only reside in PLKs, the atropisomer of formula (1) and its tartrate salts should exhibit much greater selectivity (and hence fewer unwanted side effects due to off-target kinase inhibition) than compounds which are ATP-competitive kinase inhibitors.
  • a further advantage of inhibiting the PBD domain rather than the catalytic domain is that this may result in a reduced tendency to induce drug resistance compared to PLK1 inhibitors that inhibit the catalytic domain.
  • Atropisomer of formula (1) and its tartrate salts may be effective in exploiting weaknesses in cellular pathways as a result of constitutively activating KRAS mutants and therefore may be useful for the treatment of diseases and conditions mediated by modulation of KRAS.
  • KRAS mutations are found at high rates in leukaemias, colon cancer, pancreatic cancer and lung cancer.
  • Atropisomer of formula (1) and its tartrate salts may be useful in treating cancers characterised by p53 deficiency or mutation in the TP53 gene.
  • PLK1 is believed to inhibit p53 in cancer cells. Therefore, upon treatment with PLK1 inhibitors, p53 in tumour cells should be activated and hence should induce apoptosis.
  • the activity of the atropisomer of formula (1) against KRAS mutant and p53 deficient cancers is believed to arise, at least in part, through inhibition of the C- terminal polo box domain (PBD) of PLK1 kinase as described above.
  • PPD C- terminal polo box domain
  • KRAS is known to be dependent on interaction with PLK1.
  • the atropisomer of formula (1) induces mitotic arrest with non-congressed chromosomes, a property which is believed to arise from the PLK1-PBD and PLK4-PBD inhibiting activity of the atropisomer (see Example 7C below).
  • the atropisomer induces mitotic arrest with a multipolar spindle phenotype, and causes amplification of centrioles, a well described phenotype of PLK4 inhibition (Lei 2018, Cell Death & Disease 9, 1066; Kawakami, PNAS 2018, 115(8) 1913- 18). These phenotypes are believed to arise from the PLK4-PBD inhibiting activity of the atropisomer of formula (1).
  • Example 7A A primary screen for anticancer activity, which makes use of a cancer cell line (LI87MG, human brain (glioblastoma astrocytoma)), is described in Example 7A below.
  • the data obtained demonstrate that the R-atropisomer of formula (1) (atropisomer A-2) is far more active (IC50 of 0.22 pM) than the corresponding S- atropisomer (A-1) which has an IC50 of 4.6 pM against the LI87MG cell line.
  • the two atropisomers (A-2 and A-1) have also both been tested against a panel of forty eight cancer cells lines and the results are shown in Example 7B below.
  • the atropisomer of formula (1) (A-2) was more active than the atropisomer (A-1), in most cases by a factor of at least ten.
  • Example 7B demonstrate that the atropisomer of formula (1) (A-2) is active against a wide range of different cancer cell lines ranging from solid tumours such as pancreatic cancer, cancers of the large intestine and coIorectum, lung cancers, cancers of the brain and nerves, and blood cancers such as lymphoma and leukaemia.
  • solid tumours such as pancreatic cancer, cancers of the large intestine and coIorectum, lung cancers, cancers of the brain and nerves, and blood cancers such as lymphoma and leukaemia.
  • the atropisomer of formula (1) has good oral bioavailability (see Example 7G below) and has good brain exposure when administered orally (Example 7G). Accordingly, the composition of matter or atropisomer of the invention should be useful in treating brain cancers such as gliomas and glioblastomas.
  • the atropisomer of formula (1) will be useful in the treatment of a wide range of cancers (and their benign counterparts), for example the cancers set out in the embodiments below. Accordingly, in further embodiments (Embodiments 3.1 to 3.25), the invention provides:
  • tumours of epithelial origin adenomas and carcinomas of various types including adenocarcinomas, squamous carcinomas, transitional cell carcinomas and other carcinomas
  • tumours of epithelial origin adenomas and carcinomas of various types including adenocarcinomas, squamous carcinomas, transitional cell carcinomas and other carcinomas
  • carcinomas of the bladder and urinary tract breast, gastrointestinal tract (including the oesophagus, stomach (gastric), small intestine, colon, rectum and anus), liver (hepatocellular carcinoma), gall bladder and biliary system, exocrine pancreas, kidney, lung (for example adenocarcinomas, small cell lung carcinomas, non-small cell lung carcinomas, bronchioalveolar carcinomas and mesotheliomas), head and neck (for example cancers of the tongue, buccal cavity, larynx, pharynx,
  • tumours of epithelial origin adenomas and carcinoma
  • leukaemias, lymphomas and premalignant haematological disorders and disorders of borderline malignancy including haematological malignancies and related conditions of lymphoid lineage (for example acute lymphocytic leukaemia [ALL], chronic lymphocytic leukaemia [CLL], B-cell lymphomas such as diffuse large B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt’s lymphoma, mantle cell lymphoma, T-cell lymphomas and leukaemias, natural killer [NK] cell lymphomas, Hodgkin’s lymphomas, hairy cell leukaemia, monoclonal gammopathy of uncertain significance, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorders), and haematological malignancies and related conditions of myeloid lineage (for example acute myelogenous leukaemia [AML], chronic myelogenous leukaemia [CML], chronic mye
  • 3.4A A tartrate salt or a composition of matter according to any one of Embodiments 1.1 to 1.19 for use in treating a cancer, optionally in combination with another therapeutic agent or treatment (e.g. an anticancer agent or therapy), where the cancer is selected from pancreatic cancers, cancers of the large intestine and coIorectum, lung cancers, cancers of the brain and nerves, blood cancers (such as lymphoma and leukaemia), prostate cancers and breast cancers.
  • another therapeutic agent or treatment e.g. an anticancer agent or therapy
  • Another therapeutic agent or treatment e.g. an anticancer agent or therapy
  • the cancer is selected from pancreatic cancer, cancers of the large intestine and coIorectum, lung cancers, cancers of the brain and nerves, and blood cancers such as lymphoma and leukaemia.
  • 3.6 A tartrate salt or a composition of matter according to any one of Embodiments 1.1 to 1.19 for use in treating a cancer, optionally in combination with another therapeutic agent or treatment (e.g. an anticancer agent or therapy), where the cancer is selected from gliomas and
  • another therapeutic agent or treatment e.g. an anticancer agent or therapy
  • tumours of epithelial origin selected from breast cancers, gastrointestinal tract cancers, exocrine pancreas cancers, lung and prostate cancers;
  • haematological malignancies selected from B-cell lymphomas such as diffuse large B-cell lymphoma [DLBCL], Burkitt’s lymphoma, mantle cell lymphoma, multiple myeloma, acute myelogenous leukaemia [AML], chronic myelogenous leukaemia [CML], and myelodysplastic syndrome;
  • B-cell lymphomas such as diffuse large B-cell lymphoma [DLBCL], Burkitt’s lymphoma, mantle cell lymphoma, multiple myeloma, acute myelogenous leukaemia [AML], chronic myelogenous leukaemia [CML], and myelodysplastic syndrome;
  • tumours of mesenchymal origin selected from osteosarcomas and rhabdomyosarcomas;
  • tumours of the central or peripheral nervous system selected from gliomas, glioblastomas and ependymomas;
  • another therapeutic agent or treatment e.g. an anticancer agent or therapy
  • another therapeutic agent or treatment e.g. an anticancer agent or therapy
  • Embodiment 3.11 A tartrate salt or a composition of matter for use according to Embodiment 3.10 wherein the cancer is as defined in any one of Embodiments 3.4 to 3.7.
  • another therapeutic agent or treatment e.g. an anticancer agent or therapy
  • Embodiment 3.13 A tartrate salt or a composition of matter for use according to Embodiment 3.12 wherein the cancer is as defined in any one of Embodiments 3.4 to 3.7.
  • another therapeutic agent or treatment e.g. an anticancer agent or therapy
  • a method of treating a subject e.g. a mammalian subject such as human
  • a cancer as defined in any one of Embodiments 3.4 to 3.16
  • which method comprises administering to the subject a therapeutically effective amount of a tartrate salt or a composition of matter according to any one of Embodiments 1.1 to 1.19, optionally in combination with another therapeutic agent or treatment (e.g. an anticancer agent or therapy).
  • another therapeutic agent or treatment e.g. an anticancer agent or therapy.
  • a method of inhibiting PLK1-PBD which method comprises bringing an effective inhibiting amount of a tartaric acid salt or a composition of matter according to any one of Embodiments 1.1 to 1.19 into contact with the PLK1-PBD.
  • a method of inhibiting PLK4-PBD which method comprises bringing an effective inhibiting amount of a tartaric acid salt or a composition of matter according to any one of Embodiments 1.1 to 1.19 into contact with the PLK4-PBD.
  • 3.24 A method of inhibiting PLK1-PBD and PLK4-PBD, which method comprises bringing an effective inhibiting amount of a tartaric acid salt or a composition of matter according to any one of Embodiments 1.1 to 1.19 into contact with the PLK1-PBD and PLK4-PBD. 3.25 A method according to any one of Embodiments 3.22 to 3.24 wherein the effective inhibiting amount of a tartaric acid salt or a composition of matter according to any one of Embodiments 1.1 to 1.19 is brought into contact with the PLK1-PBD and/or PLK4-PBD in vivo, for example in a mammalian subject such as a human subject.
  • a patient Prior to administration of a tartaric acid salt or a composition of matter according to any one of Embodiments 1.1 to 1.19, a patient may be screened to determine whether a cancer from which the patient is or may be suffering is one which is characterised by elevated levels of PLK1 and/or PLK4 kinase and which would therefore be would be susceptible to treatment with a compound having activity against PLK1 and/or PLK4 kinase.
  • a biological sample taken from a patient may be analysed to determine whether a cancer, that the patient is or may be suffering from is one which is characterised by a genetic abnormality or abnormal protein expression which leads to up-regulation of PLK1 and/or PLK4 kinase.
  • up-regulation includes elevated expression or over-expression, including gene amplification (i.e. multiple gene copies) and increased expression by a transcriptional effect, and hyperactivity and activation, including activation by mutations.
  • the patient may be subjected to a diagnostic test to detect a marker characteristic of upregulation of PLK1 and/or PLK4 kinase.
  • diagnosis includes screening.
  • marker we include genetic markers including, for example, the measurement of DNA composition to identify mutations of PLK1.
  • marker also includes markers which are characteristic of up-regulation of PLK1 and/or PLK4, including enzyme activity, enzyme levels, enzyme state (e.g. phosphorylated or not) and mRNA levels of the aforementioned proteins.
  • Tumours with upregulation of PLK1 and/or PLK4 kinase may be particularly sensitive to PLK1 inhibitors. Tumours may preferentially be screened for upregulation of PLK1 and/or PLK4.
  • the patient may be subjected to a diagnostic test to detect a marker characteristic of up-regulation of PLK1 and/or PLK4.
  • the diagnostic tests are typically conducted on a biological sample selected from tumour biopsy samples, blood samples (isolation and enrichment of shed tumour cells), stool biopsies, sputum, chromosome analysis, pleural fluid and peritoneal fluid. Methods of identification and analysis of mutations and up-regulation of proteins are known to a person skilled in the art. Screening methods could include, but are not limited to, standard methods such as reverse-transcriptase polymerase chain reaction (RT-PCR) or in-situ hybridisation.
  • RT-PCR reverse-transcriptase polymerase chain reaction
  • telomere amplification is assessed by creating a cDNA copy of the mRNA followed by amplification of the cDNA by PCR.
  • Methods of PCR amplification, the selection of primers, and conditions for amplification, are known to a person skilled in the art.
  • Nucleic acid manipulations and PCR are carried out by standard methods, as described for example in Ausubel, F.M. et al., eds. Current Protocols in Molecular Biology, 2004, John Wiley & Sons Inc., or Innis, M.A. et-al., eds. PCR Protocols: a guide to methods and applications, 1990, Academic Press, San Diego.
  • FISH fluorescence in-situ hybridisation
  • in situ hybridization comprises the following major steps: (1) fixation of tissue to be analyzed; (2) pre-hybridization treatment of the sample to increase accessibility of target nucleic acid, and to reduce nonspecific binding; (3) hybridization of the mixture of nucleic acids to the nucleic acid in the biological structure or tissue; (4) post-hybridization washes to remove nucleic acid fragments not bound in the hybridization, and (5) detection of the hybridized nucleic acid fragments.
  • the probes used in such applications are typically labelled, for example, with radioisotopes or fluorescent reporters.
  • Preferred probes are sufficiently long, for example, from about 50, 100, or 200 nucleotides to about 1000 or more nucleotides, to enable specific hybridization with the target nucleic acid(s) under stringent conditions.
  • Standard methods for carrying out FISH are described in Ausubel, F.M. et al., eds. Current Protocols in Molecular Biology, 2004, John Wiley & Sons Inc and Fluorescence In Situ Hybridization: Technical Overview by John M. S. Bartlett in Molecular Diagnosis of Cancer, Methods and Protocols, 2nd ed.; ISBN: 1-59259-760-2; March 2004, pps. 077-088; Series: Methods in Molecular Medicine.
  • the protein products expressed from the mRNAs may be assayed by immunohistochemistry of tumour samples, solid phase immunoassay with microtiter plates, Western blotting, 2-dimensional SDS-polyacrylamide gel electrophoresis, ELISA, flow cytometry and other methods known in the art for detection of specific proteins. Detection methods would include the use of site specific antibodies. The skilled person will recognize that all such well-known techniques for detection of up-regulation of PLK1 and/or PLK4 kinase could be applicable in the present case.
  • a patient may be screened to determine whether a cancer from which the patient is or may be suffering is one which is characterised by mutated KRAS and which would therefore be would be susceptible to treatment with a compound having activity against cancer cells carrying a mutant KRAS.
  • a biological sample taken from a patient may be analysed to determine whether a cancer, that the patient is or may be suffering from is one which is characterised by a presence of mutant KRAS.
  • the patient may be subjected to a diagnostic test to detect mutations in at codons 12, 13, 61 or mixtures thereof in the KRAS protein.
  • diagnostic tests for mutant KRAS include the cobas ® KRAS Mutation Test from Roche Molecular Systems, Inc and therascreen KRAS RGQ PCR Kit from Qiagen Manchester, Ltd.
  • Tumours with mutant KRAS may be particularly sensitive to PLK1 and/or PLK4 inhibitors.
  • Methods of identification and analysis of mutations and up-regulation of proteins are known to a person skilled in the art. Screening methods could include, but are not limited to, standard methods such as reverse-transcriptase polymerase chain reaction (RT-PCR) or in-situ hybridisation as described above.
  • RT-PCR reverse-transcriptase polymerase chain reaction
  • in-situ hybridisation as described above.
  • the invention provides: 3.26 A tartaric acid salt or a composition of matter according to any one of Embodiments 1.1 to 1.19 for use in the treatment of a cancer in a subject (e.g. a human subject) who has been screened and has been determined as suffering from a cancer which is characterised by elevated levels of PLK1 kinase (e.g. PLK1 overexpression).
  • a subject e.g. a human subject
  • PLK1 kinase e.g. PLK1 overexpression
  • a subject e.g. a human subject
  • PLK4 kinase e.g. PLK4 overexpression
  • a subject e.g. a human subject
  • PLK4 kinase e.g. PLK1 and PLK4 overexpression
  • a subject e.g. a human subject
  • a method for the diagnosis and treatment of a disease state or condition e.g. a cancer, for example a cancer as defined in any one of Embodiments 3.4 to 3.16 characterised by the presence of a mutated form of KRAS which method comprises (i) screening a subject (e.g.
  • a human subject to determine whether a disease or condition from which the subject is or may be suffering is one which would be susceptible to treatment with a compound having activity against KRAS; and (ii) where it is indicated that the disease or condition from which the subject is thus susceptible, thereafter administering to the subject a therapeutically effective amount of a tartaric acid salt or a composition of matter according to any one of Embodiments 1.1 to 1.19.
  • a method for the treatment of a disease state or condition characterised by the presence of a mutated form of KRAS, which method comprises administering a therapeutically effective amount of a tartaric acid salt or a composition of matter according to any one of Embodiments 1.1 to 1.19 to a subject (e.g. a human subject) who has been screened and has been determined as suffering from, or being at risk of suffering from, a disease or condition which would be susceptible to treatment with a compound having activity against KRAS.
  • a disease state or condition e.g. a cancer, for example a cancer as defined in any one of Embodiments 3.4 to 3.16 characterised by the presence of a mutated form of KRAS
  • a subject e.g. a human subject
  • the tartaric acid salt or a composition of matter according to any one of Embodiments 1.1 to 1.19 are typically administered to patients in the form of a pharmaceutical composition. Accordingly, in another Embodiment of the invention (Embodiment 4.1), the invention provides a pharmaceutical composition comprising a tartaric acid salt or a composition of matter according to any one of Embodiments 1.1 to 1.19 and a pharmaceutically acceptable excipient.
  • a pharmaceutical composition according to Embodiment 4.1 which comprises from approximately 1% (w/w) to approximately 95% (w/w) of a tartaric acid salt or a composition of matter according to any one of Embodiments 1.1 to 1.19 and from 99% (w/w) to 5% (w/w) of a pharmaceutically acceptable excipient or combination of excipients and optionally one or more further therapeutically active ingredients.
  • a pharmaceutical composition according to Embodiment 4.2 which comprises from approximately 5% (w/w) to approximately 90%,% (w/w) of a composition of tartaric acid salt or a composition of matter according to any one of Embodiments 1.1 to 1.19 and from 95% (w/w) to 10% of a pharmaceutically excipient or combination of excipients and optionally one or more further therapeutically active ingredients.
  • a pharmaceutical composition according to Embodiment 3.3 which comprises from approximately 10% (w/w) to approximately 90%,% (w/w) of a tartaric acid salt or a composition of matter according to any one of Embodiments
  • a pharmaceutical composition according to Embodiment 4.4 which comprises from approximately 20% (w/w) to approximately 90%,% (w/w) of a tartaric acid salt or a composition of matter according to any one of Embodiments
  • a pharmaceutical composition according to Embodiment 3.5 which comprises from approximately 25% (w/w) to approximately 80%,% (w/w) of a tartaric acid salt or a composition of matter according to any one of Embodiments
  • compositions of the invention can be in any form suitable for oral, parenteral, topical, intranasal, intrabronchial, ophthalmic, otic, rectal, intra- vaginal, or transdermal administration.
  • compositions are intended for parenteral administration, they can be formulated for intravenous, intramuscular, intraperitoneal, subcutaneous administration or for direct delivery into a target organ or tissue by injection, infusion or other means of delivery.
  • Pharmaceutical dosage forms suitable for oral administration include tablets, capsules, caplets, pills, lozenges, syrups, solutions, sprays, powders, granules, elixirs and suspensions, sublingual tablets, sprays, wafers or patches and buccal patches.
  • the invention provides: 4.7 A pharmaceutical composition according to any one of Embodiments 4.1 to 4.6 which is suitable for oral administration.
  • a pharmaceutical composition according to Embodiment 4.7 which is selected from tablets, capsules, caplets, pills, lozenges, syrups, solutions, sprays, powders, granules, elixirs and suspensions, sublingual tablets, sprays, wafers or patches and buccal patches.
  • a pharmaceutical composition according to Embodiment 4.8 which is selected from tablets and capsules.
  • a pharmaceutical composition according to Embodiment 4.10 which is formulated for intravenous, intramuscular, intraperitoneal, subcutaneous administration or for direct delivery into a target organ or tissue by injection, infusion or other means of delivery.
  • Embodiment 4.12 A pharmaceutical composition according to Embodiment 4.11 which is a solution or suspension for injection or infusion.
  • compositions e.g. as defined in any one of Embodiments 4.1 to 4.12 containing the tartaric acid salt or a composition of matter according to any one of Embodiments 1.1 to 1.19 can be formulated in accordance with known techniques, see for example, Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, USA.
  • tablet compositions can contain a unit dosage of active compound together with an inert diluent or carrier such as a sugar or sugar alcohol, e.g.; lactose, sucrose, sorbitol or mannitol; and/or a non-sugar derived diluent such as sodium carbonate, calcium phosphate, talc, calcium carbonate, or a cellulose or derivative thereof such as methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, and starches such as corn starch. Tablets may also contain such standard ingredients as binding and granulating agents such as polyvinylpyrrolidone, disintegrants (e.g.
  • swellable crosslinked polymers such as crosslinked carboxymethylcellulose
  • lubricating agents e.g. stearates
  • preservatives e.g. parabens
  • antioxidants e.g. BHT
  • buffering agents for example phosphate or citrate buffers
  • effervescent agents such as citrate/bicarbonate mixtures.
  • Capsule formulations may be of the hard gelatin or soft gelatin variety and can contain the active component in solid, semi-solid, or liquid form.
  • Gelatin capsules can be formed from animal gelatin or synthetic or plant derived equivalents thereof.
  • the solid dosage forms can be coated or un-coated, but typically have a coating, for example a protective film coating (e.g. a wax or varnish) or a release controlling coating.
  • a protective film coating e.g. a wax or varnish
  • the coating e.g. a Eudragit TM type polymer
  • the coating can be designed to release the active component at a desired location within the gastro-intestinal tract.
  • the coating can be selected so as to degrade under certain pH conditions within the gastrointestinal tract, thereby selectively release the composition of matter or atropisomer in the stomach or in the ileum or duodenum.
  • the drug can be presented in a solid matrix comprising a release controlling agent, for example a release delaying agent which may be adapted to selectively release the composition of matter or atropisomer under conditions of varying acidity or alkalinity in the gastrointestinal tract.
  • a release controlling agent for example a release delaying agent which may be adapted to selectively release the composition of matter or atropisomer under conditions of varying acidity or alkalinity in the gastrointestinal tract.
  • the matrix material or release retarding coating can take the form of an erodible polymer (e.g. a maleic anhydride polymer) which is substantially continuously eroded as the dosage form passes through the gastrointestinal tract.
  • an erodible polymer e.g. a maleic anhydride polymer
  • compositions for topical use include ointments, creams, sprays, patches, gels, liquid drops and inserts (for example intraocular inserts). Such compositions can be formulated in accordance with known methods.
  • compositions for parenteral administration are typically presented as sterile aqueous or oily solutions or fine suspensions, or may be provided in finely divided sterile powder form for making up extemporaneously with sterile water for injection.
  • formulations for rectal or intra-vaginal administration include pessaries and suppositories which may be, for example, formed from a shaped mouldable or waxy material containing the active compound.
  • Compositions for administration by inhalation may take the form of inhalable powder compositions or liquid or powder sprays, and can be administrated in standard form using powder inhaler devices or aerosol dispensing devices. Such devices are well known.
  • the powdered formulations typically comprise the active compound together with an inert solid powdered diluent such as lactose.
  • compositions will generally be presented in unit dosage form and, as such, will typically contain sufficient compound to provide a desired level of biological activity.
  • a composition intended for oral administration may contain from 2 milligrams to 200 milligrams of active ingredient, more usually from 10 milligrams to 100 milligrams, for example, 12.5 milligrams, 25 milligrams and 50 milligrams.
  • the active compound (tartaric acid salt or a composition of matter according to any one of Embodiments 1.1 to 1.19) will be administered to a patient in need thereof (for example a human or animal patient) in an amount sufficient to achieve the desired therapeutic effect: e.g. an effect as set out in Embodiments 3.1 to 3.34 above.
  • the tartaric acid salt or a composition of matter according to any one of Embodiments 1.1 to 1.19 will generally be administered to a subject in need of such administration, for example a human or animal patient, preferably a human.
  • the tartaric acid salt or a composition of matter according to any one of Embodiments 1.1 to 1.19 will typically be administered in amounts that are therapeutically or prophylactically useful and which generally are non-toxic.
  • the benefits of administering compounds of the invention may outweigh the disadvantages of any toxic effects or side effects, in which case it may be considered desirable to administer compounds in amounts that are associated with a degree of toxicity.
  • a typical daily dose of the tartaric acid salt or a composition of matter according to any one of Embodiments 1.1 to 1.19 can be in the range from 0.025 milligrams to 5 milligrams per kilogram of body weight, for example up to 3 milligrams per kilogram of bodyweight, and more typically 0.15 milligrams to 5 milligrams per kilogram of bodyweight although higher or lower doses may be administered where required.
  • a typical daily dose of the tartaric acid salt or a composition of matter according to any one of Embodiments 1.1 to 1.19 can be in the range from 0.025 milligrams to 50 milligrams per kilogram of body weight, for example up to 30 milligrams per kilogram of bodyweight, and more typically 0.15 milligrams to 50 milligrams per kilogram (e.g. from 0.5 milligrams to 30 milligrams per kilogram) of bodyweight although higher or lower doses may be administered where required.
  • an initial starting dose of 12.5 mg may be administered 2 to 3 times a day.
  • the dosage can be increased by 12.5 mg a day every 3 to 5 days until the maximal tolerated and effective dose is reached for the individual as determined by the physician.
  • a once weekly dosing schedule may comprise an initial starting dose of 0.5-1.5 mg/kg (e.g. 1 mg/kg) in week one followed in week two and subsequent weeks by escalating doses (e.g. two or three times the previous dose for up to three, four or five dose escalations) up to a maximum dosage consistent with therapeutic effect and tolerability to the subject.
  • escalating doses e.g. two or three times the previous dose for up to three, four or five dose escalations
  • a starting dose of 1 mg/kg may be administered followed by an increased dose in the second week of 3 mg/kg, 9 mg/kg in the third week and 27 mg/kg in the fourth week.
  • the quantity of compound administered will be commensurate with the nature of the disease or physiological condition being treated and the therapeutic benefits and the presence or absence of side effects produced by a given dosage regimen, and will be at the discretion of the physician.
  • the tartaric acid salt or a composition of matter according to any one of Embodiments 1.1 to 1.19 will be useful either as sole chemotherapeutic agents or, more usually, in combination therapy with chemotherapeutic agents or radiation therapy in the prophylaxis or treatment of a range of proliferative disease states or conditions. Examples of such disease states and conditions are set out above.
  • chemotherapeutic agents or other treatments that may be co-administered with the tartaric acid salt or a composition of matter according to any one of Embodiments 1.1 to 1.19 include:
  • Topoisomerase I inhibitors e.g. irinotecan
  • Antimetabolites (e.g. cytarabine or gemcitabine)
  • Tubulin targeting agents e.g. paclitaxel
  • EGFR inhibitors e.g. gefitinib or afatinib
  • mTOR inhibitors e.g. everolimus
  • PI3K pathway inhibitors e.g. PI3K, PDK1
  • Alkylating Agents e.g. temozolomide
  • hypoxia triggered DNA damaging agents e.g. tirapazamine
  • Anti cd20 antibodies e.g. rituximab
  • HER2 small molecule inhibitors e.g. lapatinib or afatinib
  • Bcr-Abl tyrosine-kinase inhibitors e.g. imatinib
  • CDK4/6 inhibitor e.g. palbociclib
  • Inhibitors of immune checkpoint blockade signalling components including PD1, PDL-1 and CTLA4;
  • KRAS blocking drugs including those against specific mutations such as G12C (e.g. sotorasib);
  • Bcl2 inhibitors e.g. venetoclax, sabutoclax or obatoclax
  • chemotherapeutic agents or other treatments that may be co-administered with the tartaric acid salt or a composition of matter according to any one of Embodiments 1.1 to 1.19 may be selected from:
  • Topoisomerase I inhibitors e.g. irinotecan
  • Antimetabolites (e.g. cytarabine or gemcitabine)
  • Tubulin targeting agents e.g. paclitaxel
  • EGFR inhibitors e.g. gefitinib or afatinib
  • mTOR inhibitors e.g. everolimus
  • Alkylating Agents e.g. temozolomide
  • Anti cd20 antibodies e.g. rituximab
  • Inhibitors of immune checkpoint blockade signalling components including PD1, PDL-1 and CTLA4; • KRAS blocking drugs, including those against specific mutations suchas G12C (e.g. sotorasib);
  • Bcl2 inhibitors e.g. venetoclax, sabutoclax or obatoclax
  • the invention provides:
  • a pharmaceutical combination comprising a tartaric acid salt or a composition of matter according to any one of Embodiments 1.1 to 1.19 and another therapeutically active agent.
  • a pharmaceutical composition comprising a tartaric acid salt or a composition of matter according to any one of Embodiments 1.1 to 1.19, another therapeutically active agent and at least one pharmaceutically acceptable excipient.
  • a method of treatment of a subject suffering from a cancer which method comprises the administration to the subject of a therapeutically effective amount of a pharmaceutical combination according to any one of Embodiments 5.1 to 5.5.
  • Figure 1 is a schematic diagram illustrating the R/S classification system for atropisomers.
  • Figure 2 is a depiction of the three dimensional structure of 2,4-[5-(4-chlorophenyl)- 1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2-(dimethylamino)-ethyl]benzamide atropisomer A-2 as determined by single crystal X-ray crystallographic studies.
  • Figure 3 is a schematic stereochemical illustration of the two atropisomers A-1 (S) and A-2 (F?) and the basis for assigning their stereochemical structures using the Cahn-lngold-Prelog (CIP) sequence rules.
  • Figure 4 is an X-ray powder diffraction spectrum for atropisomer A-2 free base.
  • Figure 5 is an X-ray powder diffraction spectrum for atropisomer A-2 Tartrate Pattern A salt (bottom line) and Pattern B salt (top and middle lines)
  • Figure 6 illustrates the thermal profile for atropisomer A-2 free base and shows a differential scanning calorimetry plot (line 6A) and a thermo-gravimetric analysis plot (line 6B).
  • Figure 7 illustrates the thermal profile for atropisomer A-2 Tartrate Pattern A salt and shows a differential scanning calorimetry plot (line 7A) and a thermo- gravimetric analysis plot (line 7B).
  • Figure 8 illustrates the thermal profile for atropisomer A-2 Tartrate Pattern B salt and shows a differential scanning calorimetry plot (line 8A) and a thermo- gravimetric analysis plot (line 8B).
  • Figure 9 is a plot of weight change versus relative humidity in Gravimetric Vapour Sorption studies carried out on atropisomer A-2 Tartrate Pattern B salt.
  • Figure 10 is a bar chart showing the proportions of different observed mitotic phenotypes (non-congressed chromosomes, multipolar spindles/abnormal cytokinesis, monopolar spindles, normal prometaphase, normal metaphase produced after) after treating LI87MG cells with 0.03 pM concentrations of either of atropisomer A-1 or atropisomer A-2.
  • Figure 11 is a bar chart showing the numbers of centrioles present in HeLa cells after treatment with 0.02 pM concentrations of either of atropisomer A-1 or atropisomer A-2.
  • Figure 12 is a plot of blood plasma concentrations against time following oral and i.v. dosing to mice of atropisomer A-2. The lower line, extending as far as 24 hours, is the line for the 2 mg/kg i.v. dose. The other line is for the 10 mg/kg p.o. dose.
  • Figure 13 is a plot of blood plasma and brain concentrations against time following oral dosing (10 mg/kg) to mice of atropisomer A-2.
  • the upper line shows the brain concentrations while the lower line shows the plasma concentrations.
  • Figure 14 is a plot of tumour volume versus time in male athymic nude mice in a LI87MG subcutaneous xenograft model after administration of atropisomer A-2.
  • Figure 15 is a graphic comparison of bioluminescent signal linked to tumour growth in male athymic nude mice in a U87-Luc orthotopic xenograft model after administration of atropisomer A-2.
  • Figure 16 is a plot of tumour volume versus time in male athymic nude mice in an HCT 116 subcutaneous xenograft model after administration of atropisomer A-2.
  • LCMS was carried out on LIPLC AQLIITY with PDA photodiode array detector and QDa mass detector.
  • the column used was a C18, 2.1 x 50mm, 1.9 pm.
  • the sample was prepared in methanokacetonitrile to achieve an approximate concentration of 250 ppm.
  • HPLC Method 1 HPLC analysis was carried out on an Agilent Technologies 1100/1200 series
  • HPLC system HPLC system.
  • the column used was an ACE 3 C18; 150 x 4.6mm, 3.0pm particle size (Ex: Hichrom, Part number: ACE-111-1546).
  • the flow rate was 1.0 mL/min.
  • Mobile phase A was watertrifluoroacetic acid (100:0.1 %) and mobile phase B was acetonitrile:trifluoroacetic acid (100:0.1%).
  • the injection volume was 5 pL and the following gradient was used:
  • Chiral HPLC was analysis was carried out on an Agilent Technologies 1200 series HPLC system.
  • the column used was a CHIRAL PAK IG, 250 x 4.6 mm, 5 pm.
  • the column flow rate was 1.0 mL/min and the mobile phase was: (A) 0.1 % v/v DEA in n-heptane and (B) IPA:MeOH (70:30).
  • the injection volume was 25 pL. Samples were prepared in IPA:MeOH to achieve an approximate concentration of 250 ppm and with the following isocratic method:
  • Chiral HPLC was carried out on an Agilent Technologies 1200 series HPLC system.
  • the column used was a CHIRAL PAK IG, 250 x 4.6mm, 5 pm.
  • the column flow rate was 1.0 mL/min and the mobile phase was: (A) 0.1% v/v DEA in n-heptane and (B) IPA: MEOH (70:30).
  • the injection volume was 10 pL.
  • Samples were prepared in IPA:MeCN to achieve an approximate concentration of 250 ppm and with the following isocratic method:
  • Chiral HPLC was analysis was carried out on an Agilent Technologies 1100/1200 series HPLC system.
  • the column used was a CHIRALPAK AD-H; 250 x 4.6mm, 5.0pm.
  • the column flow rate was 1.0 mL/min and the mobile phase was: Hexane:EtOH:TFA (90:10:0.1%).
  • the injection volume was 5 pL. Samples were prepared in 100% EtOH to achieve an approximate concentration of 0.5 mg/mL.
  • Chiral HPLC was analysis was carried out on an Agilent Technologies 1100/1200 series HPLC system.
  • the column used was a CHIRALPAK IA; 250 x 4.6mm, 5.0pm.
  • the column flow rate was 1.0 mL/min and the mobile phase was: Hexane:EtOH:Ethanolamine (90:10:0.1%).
  • the injection volume was 5 pL.
  • the atropisomers were isolated using one of the following preparative chiral HPLC methods.
  • Preparative chiral HPLC was carried out using a CHIRALPAK IG SFC, 21 x 250 mm, 5pm column, eluting with (A) 0.1 % DEA in heptane and (B) I PA as mobile phase, with the flow rate of 30 mL/min and the following isocratic system:
  • Preparative chiral HPLC was carried out using a CHIRALPAK IG SFC column, 21 x 250 mm, 5pm eluting with (A) 0.1% DEA in heptane and (B) IPA:MeOH (90:10) as mobile phase and a flow rate of 22 mL/min and with the following isocratic system was used for the elution:
  • Step 1 4-r4-(4-chlorophenyl)-4-oxo-butanoyl1benzonitrile (6)
  • Zinc chloride (30.5 g, 223 mmol) was heated to melting under vacuum then cooled to room temperature. Toluene (100 mL), tert-butanol (16.5 mL, 172 mmol) and triethylamine (24 mL, 172 mmol) were added and the mixture was stirred at room temperature for 2 hours under a nitrogen atmosphere at which point the zinc chloride had fully dissolved. 4-Cyanoacetophenone (25 g, 172 mmol) and 4- chlorophenacylbromide (40.2 g, 172 mmol) were added and the reaction mixture was stirred at room temperature for 48 hours.
  • Step 2 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl) phenyl)-1 H-pyrrol-2-yl) benzonitrile (7)
  • Step 3 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl) phenyl)-1 H-pyrrol-2-yl) benzoic acid (8)
  • Step 4b 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl) phenyl)-1 H-pyrrol-2-yl)-N-(2-
  • Step 5b Separation of Atropisomers
  • the atropisomers of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]- N-[2-(dimethylamino)ethyl]benzamide were resolved by chiral HPLC using preparative Chiral HPLC Method 1.
  • Peak 1 Example A-1 , 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2- yl]-N-[2 (dimethylamino)ethyl]benzamide - atropisomerl (0.3 g, 0.58 mmol, 38%, >99% ee), and:
  • the compounds can also be isolated as their hydrochloride salts.
  • Atropisomer A-1 (S)-4-f5-(4-chlorophenyl)-1-r2-(trifluoromethyl)phenyllpyrrol-2-yl1- N-f2 (dimethylamino)ethyllbenzamide hydrochloride salt
  • Peak 1 (0.31g, 0.606 mmol) from Example 1, Step 5b was further purified by stirring in HPLC grade water (30 mL) followed by sonication for 10 min and extraction with ethyl acetate (3 x 30 mL). The combined organic layers were dried (Na2SC>4), filtered and concentrated under reduced pressure followed by lyophilisation to afford an amorphous solid (0.290 g, 0.567 mmol, 94%) which was dissolved in dichloromethane (7.12 mL). The resulting solution was cooled to 0°C and 4N HCI in dioxane (1.42 mL) was added. The reaction mixture was stirred at room temperature for 3 hours. The mixture was concentrated and dried under high vacuum. Purification by trituration using diethyl ether (10 mL) and lyophilisation afforded the title compound (0.3 g, 0.56 mmol, 98 %) as an off-white solid.
  • Atropisomer A-2 (R)-4-r5-(4-chlorophenyl)-1-r2-(trifluoromethyl)phenyllpyrrol-2-yl1- N-f2 (dimethylamino)ethyllbenzamide hydrochloride salt
  • the hydrochloride salt of atropisomer A-2 was prepared using the same method as was used for atropisomer A-1 starting from peak 2 to afford the title compound (0.31 g, 0.56 mmol, 99%), an off-white solid.
  • Atropisomer A-2 Single crystal X-ray crystallographic analysis of atropisomer A-2 (see Example 3 below) indicated that atropisomer A-2 is the R-isomer (Compound (1)) and hence atropisomer A-1 must be the S-isomer.
  • Atropisomer A-2 free base was prepared, and a single crystal was subjected to X- ray crystallographic studies as described below.
  • the data were collected and processed using CrysAlisPro software and the structure was solved with the SheIXT (Sheldrick, 2015) structure solution program using the Intrinsic Phasing solution method and by using Olex2 (Dolomanov et al., 2009) as the graphical interface.
  • the model was refined with version 2018/3 of ShelXL-2018/3 (Sheldrick, 2018) using Least Squares minimisation.
  • the crystal structure was found to be monoclinic and was assigned the space group P21 (# 4). All non-hydrogen atoms were refined anisotropically. Hydrogen atom positions were calculated geometrically and refined using the riding model.
  • Atropisomer A-2 is believed to have the R configuration as shown in Figures 2 and 3 and can therefore be named as (F?)-4-[5- (4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]-N-[2 (dimethylamino)- ethyl]benzamide.
  • Atropisomer A-2 free base (904.2 mg) was suspended in acetone (9.042 mL, 10 vols) and stirred at 25 °C for 40 minutes. When the solution was free of visible particulates, it was split into 12 equal aliquots (603 pL), giving an approximate active content of 60.3 mg per sample.
  • Atropisomer A-2 (749.8 mg) was suspended in isopropyl acetate (15 mL, 20 vols) and the suspension was heated to 40°C with agitation. When the solution was free of visible particulates, it was split into 12 equal aliquots (1 mL), giving an approximate active content of 50 mg per sample. An aliquot of 195.3 pL of a 1 M solution of atropisomer A-2 in ethanol was added to an aliquot of the free base solution at 40°C. The resulting mixture was cooled to 25°C at a cooling rate of approximately 10°C/hour. A white suspension formed and the resulting solids were then isolated by filtration and dried in vacuo at 40 °C for ca. 18 hours. The resulting salt was labelled as Tartrate Pattern B.
  • Method 1 was repeated, except that atropisomer A-2 (913.9 mg) was initially suspended in 2-methyl-tetrahydrofuran (15 mL, 20 vols), (9.139 mL, 10 vols) and stirred at 25 °C for ca. 40 minutes, and then a 250 pL (1.05 eq) aliquot of 1 M tartaric acid in ethanol was added to an aliquot (609 pL) of the A-2 free base solution, to give Tartrate Pattern A salt.
  • 2-methyl-tetrahydrofuran 15 mL, 20 vols
  • 9.139 mL, 10 vols stirred at 25 °C for ca. 40 minutes
  • a 250 pL (1.05 eq) aliquot of 1 M tartaric acid in ethanol was added to an aliquot (609 pL) of the A-2 free base solution, to give Tartrate Pattern A salt.
  • Atropisomer A-2 free base (521.5 mg) was weighed into a glass vial and charged with isopropyl acetate (20 vols, 10.430 mL). The mixture was heated to 40 °C and stirred for 15 minutes to give a clear solution. The solution was then charged with tartaric acid (1.05 eq, 162.5 mg) dissolved in 3 mL of tetrahydrofuran. The resulting mixture was seeded with atropisomer A-2. tartrate pattern B, which caused the salt to immediately precipitate at 40 °C forming a mobile suspension. The mixture was cooled to 25 °C and stirred for 20 hours. The resulting solid was isolated by filtration and dried at 40 °C in vacuo to afford the atropisomer A-2 Tartrate Pattern B salt in 84% yield.
  • Atropisomer A-2 free base (10.0497 g) was weighed into a Buchi flask and charged with isopropyl acetate (20 vols, 200 ml). The mixture was heated to 40 °C to afford a clear solution, free of particulates, and stirred for 30 minutes.
  • the solution was charged with tartaric acid (3.1954 g, 1.08 eq.) dissolved in tetra hydrofuran (50 mL), the acid being was added in portions as follows: 15 mL at 40 °C; seeded with atropisomer A-2 Tartrate Pattern B salt and stirred for 30 minutes; 10 mL and stirred for 1 hour; 10 mL and stirred for 30 minutes; 15 mL and stirred for 30 minutes.
  • Atropisomer A-2 free base (36.79 g) was weighed into a flask and charged with butanol (282.57 ml, 7.68 vols). The mixture was heated to 80 °C (pale yellow, hazy solution) and stirred for 30 minutes before clarification into a Mya* vessel, pre-heated at 80 °C. The solution was then charged with L-(+)-tartaric acid (1.023 eq, 11.0806 g) as a solution in water (11.77 ml, 0.32 vols of the initial API charge). The addition was made dropwise at 80 °C with clarification of the acid solution.
  • the Radley’s Mya4 Reaction Station is a 4-zone reaction station with magnetic and overhead stirring capabilities and a temperature range of -30 to 180 °C on 2 to 400 mL scale mixtures. The reaction conditions required were programmed via the Mya 4 Control Pad. Characterisation of the atropisomer A-2 tartrate salts
  • the tartrate salts were characterised using X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), gravimetric solubility tests and gravimetric vapour sorption tests using the techniques described below.
  • XRPD X-ray powder diffraction
  • DSC differential scanning calorimetry
  • TGA thermogravimetric analysis
  • gravimetric solubility tests gravimetric vapour sorption tests using the techniques described below.
  • X-Ray Powder Diffraction patterns were collected on a PANalytical diffractometer using Cu Ka radiation (45kV, 40mA), 0 - 0 goniometer, focusing mirror, divergence slit (1/2”), soller slits at both incident and divergent beam (4mm) and a PIXcel detector.
  • the software used for data collection was X’Pert Data Collector, version 2.2f and the data was presented using X’Pert Data Viewer, version 1.2d.
  • XRPD patterns were acquired under ambient conditions via a transmission foil sample stage (polyimide - Kapton, 12.7pm thickness film) under ambient conditions using a PANalytical X’Pert PRO. The data collection range was 2.994 - 35°20 with a continuous scan speed of 0.202004°s-1.
  • DSC data were collected on a PerkinElmer Pyris 6000 DSC equipped with a 45- position sample holder. The instrument was verified for energy and temperature calibration using certified indium. A predefined amount of the sample, 0.5-3.0 mg, was placed in a pin-holed aluminium pan and heated at 20 °C. min- from 30 to 350 °C or varied as experimentation dictated. A purge of dry nitrogen at 20 ml min -1 was maintained over the sample. The instrument control, data acquisition and analysis were performed with Pyris Software v11.1.1 revision H.
  • TGA Thermo-Gravimetric Analysis
  • the solubility in water of the salts was measured using a gravimetric solubility protocol.
  • Sorption isotherms were obtained using a Hiden Isochema moisture sorption analyser (model IGAsorp), controlled by IGAsorp Systems Software V6.50.48.
  • the sample was maintained at a constant temperature (25°C) by the instrument controls.
  • the humidity was controlled by mixing streams of dry and wet nitrogen, with a total flow of 250ml. min-1.
  • the instrument was verified for relative humidity content by measuring three calibrated Rotronic salt solutions (10 - 50 - 88%).
  • the weight change of the sample was monitored as a function of humidity by a microbalance (accuracy +/- 0.005 mg). A defined amount of sample was placed in a tared mesh stainless steel basket under ambient conditions.
  • a full experimental cycle typically consisted of three scans (sorption, desorption and sorption) at a constant temperature (25°C) and 10% RH intervals over a 0 - 90% range (60 minutes for each humidity level). This type of experiment should demonstrate the ability of samples studied to absorb moisture (or not) over a set of well-determined humidity ranges.
  • GVS analysis indicated a moisture content of ca. 0.3% before the first desorption. Between 80 and 90% RH there is a slightly higher increase in moisture, with the solid taking ca. 0.8% moisture.
  • the second absorption/desorption cycle shows how the moisture uptake is completely reversible, with a return to 0 wt % at at 0% RH.
  • XRPD post GVS cycling held at 0% RH and 90% RH for a minimum of 3 hours afforded anhydrous Pattern B at both RH values.
  • Solubility Atropisomer A-2 Tartrate Pattern B salt was found to be highly soluble in aqueous formulations, making it suitable for oral delivery. A concentration of >100 mg/mL was achieved in a solution of (2-hydroxypropyl)-p-cyclodextrin 20% w/v in water following continuous stirring and gentle warming to 40°C.
  • the bulk density was measured by charging Atropisomer A-2 Tartrate Pattern B salt to a 50 mL glass beaker. The compound was allowed to settle prior to weighing with approximately 40 mL volume occupied and bulk density calculated.
  • the tap density was determined by charging 6-8 mL of Atropisomer A-2 Tartrate Pattern B salt into a 10 mL graduated cylinder, following which the material was tapped and vibrated repeatedly for 15 minutes manually in a vertical and horizontal manner using a rubber mat and mallet or until a consistent bed that settled no further was achieved. The tap density was then calculated.
  • Atropisomer A-2 Tartrate Pattern B salt has a powder bulk density value of 0.55 g/mL and tap density of -0.659 g/mL.
  • the flowability of a drug powder and hence its suitability for formulating in a capsule form can be defined by its Carr’s index and Hausner ratio which can be calculated from its bulk density and tap density according to the following formulae:
  • the Carr’s index and Hausner ratio can then be converted to a flowability descriptor as set out in the table below (source: https://www.researchgate.net/figure/Specifications-for-Carrs-index-and-Hausner- ratio_tbl1 _325365029) .
  • Carr's index Flowability Hausner ratio
  • Atropisomer A-2 Tartrate Pattern B salt was assessed using two sets of storage conditions; namely 25°C ⁇ 2°C/60% RH and 40°C ⁇ 2°C/75% RH.
  • the stability protocol followed ICH guidelines.
  • Atropisomer A-2 Tartrate Pattern B salt (1 g per container) was placed into ICH rated stability cabinets at 25°C/60% RH and 40°C/75% RH with the following packing components:
  • Double polythene bags (Vendor - Armagrip; part no G01-PB-120),
  • Plastic tie strips (Vendor- Thomas & Betts; part no TY125-40-100).
  • Step 1 4-r4-(4-chlorophenyl)-4-oxo-butanoyl1benzonitrile (6)
  • Step 2 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl) phenyl)-1 H-pyrrol-2-yl) benzonitrile (7)
  • reaction mixture was cooled to room temperature and concentrated under vacuum.
  • the resulting oily residue was purified by slurring in methanol (10mL/g).
  • the solid was isolated by filtration and dried under vacuum (45°C) to afford the title compound as a yellow solid.
  • Step 3 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl) phenyl)-1 H-pyrrol-2-yl) benzoic acid (8)
  • Step 4a (R) 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl) phenyl)-1 H-pyrrol-2-yl) benzoic acid (3) by chiral resolution of (8)
  • Salt break was achieved in THF/water (2/2 vols) using 1M HCI (2.2 eq) to afford the acid which was further purified by slurry in water affording the title compound (90.52 g, salt break yield 97%, overall yield 39%, 98.06% ee).
  • Chiral HPLC with chiral HPLC method 6 showed a single atropisomer, RT 6.083 min, 99.02% area (minor atropisomer RT 7.07 min, 0.98% area).
  • Step 5a (R)-4-f5-(4-chlorophenyl)-1-r2-(trifluoromethyl)phenyllpyrrol-2-yl1-N-r2 (dimethylamino)ethyllbenzamide (1)
  • LI87MG cells were grown in their recommended growth media/supplements (ATCC). Cells were seeded at a concentration of 5000 cells per well into 96 well plates overnight at 37°C, 5% CO2. Cells were treated with relevant concentrations of test compound for 72 hours. After 72h incubation, viability was established using sulforhodamine B (SRB) colorimetric assay. Percentage viability was calculated against the mean of the DMSO treated control samples, and IC50 values for inhibition of cell growth were calculated using GraphPad Prism software by nonlinear regression (4 parameter logistic equation).
  • SRB sulforhodamine B
  • Atropisomer A-2 was a significantly more active cell growth inhibitor than atropisomer A-1 against all of the cell lines
  • Distinct mitotic phenotypes are induced following inhibition of PLK1 and PLK4 in cells. Disruption of the PBD domain of PLK1 has been demonstrated to trigger mitotic arrest with non-congressed chromosomes, a distinct phenotype from the monopolar spindle phenotype induced by ATP-competitive PLK1 inhibitors (Hanisch et al., 2006 Mol. Biol. Cell 17, 448-459). Centriole assembly is controlled by PLK4, with inhibitors inducing a multipolar spindle phenotype due to centrosome defects which results in abnormal cyokinesis (Wong et al., 2015. Science 348(6239); 1155-1160).
  • GraphPad Prism was used to plot % mitotic cells against compound concentration using log(inhibitor) vs response variable slope with least squares fitting and no constraints. From the results obtained by following the above protocol, the EC50 values and the percentages of cells in mitosis against the HeLa and LI87MG cell lines were obtained for atropsiomer A-2. The EC50 values are shown in Table 11 below.
  • Atropisomer A-2 exhibits evidence of PLK4 inhibition phenotypes on HeLa cells.
  • Atropisomer A-2 was tested on HeLa cells engineered to inducibly express wild-type or oncogenic KRasG 12 V transgenes using the FLP-in/T-Rex system (Invitrogen). Cells were plated, and then treated with or without doxycycline to induce transgene expression, and then treated with serially-diluted atropisomer A- 2. After 72 hours of incubation, cell viability was assessed using the Cell Titre Blue reagent (Promega) and a BMG Pherastar plate reader. The effect of PBD inhibition on cell viability with either wild-type or oncogenic G12V KRAS was assessed using GraphPad Prism.
  • Atropisomer A-2 binds to the PBD domains of PLK1 and PLK4 but not to the catalytic domains of PLK1 and PLK4 and should exhibit good selectivity over other kinases. This has been investigated by testing atropisomer A-2 for off-target activity against a panel of ninety-seven kinases distributed across the kinome at a concentration of 3 pM using the DiscoverX KinomeScreen assay.
  • the DiscoverX KinomeScreen assay is a site-directed competition binding assay which measures the binding affinity of a compound to a kinase, by use of a solid supported control compound which can bind or capture the kinases in solution. In the absence of a kinase-inhibitor test compound, all of the kinase will bind to the solid support. If a kinase-inhibitor test compound is added to the assay mix, the amount of kinase binding to the solid support will be reduced, the extent of reduction being dependent on the potency of the test compound as a kinase inhibitor.
  • the potencies of the test compounds against the kinases can be expressed as the percentage (Percent Control) of the kinase binding to the solid support at a given concentration of the test compound, the lower the percentage the more potent the kinase-binding capability of the test compound.
  • Percent Control 100% would indicate that the test compound does not bind to the kinase at all, since all of the kinase has bound to the solid support.
  • a Percent Control value of 0% would indicate that the test compound has bound all of the kinase since none is bound to the solid support.
  • kinase-tagged T7 phage strains were grown in parallel in 24-well blocks in an E. coli host derived from the BL21 strain.
  • Binding reactions were assembled by combining kinases, liganded affinity beads, and test compounds in 1x binding buffer (20 % SeaBlock, 0.17x PBS, 0.05 % Tween 20, 6 mM DTT). Test compounds were prepared as 40x stocks in 100% DMSO and directly diluted into the assay. All reactions were performed in polypropylene 384-well plates in a final volume of 0.02 ml.
  • the assay plates were incubated at room temperature with shaking for 1 hour and the affinity beads were washed with wash buffer (1x PBS, 0.05 % Tween 20). The beads were then re-suspended in elution buffer (1x PBS, 0.05 % Tween 20, 0.5 pM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The kinase concentration in the eluates was measured by qPCR.
  • the strength of binding of the test molecule to the kinase can be expressed as the percent control (%Ctrl)
  • the compound(s) were screened at 3000 nM concentration, and results for primary screen binding interactions are reported as '% Ctrl', where lower numbers indicate stronger hits in the matrix on the following page(s).
  • Atropisomer A-2 was evaluated in an in vivo mouse model to determine brain and plasma concentrations following p.o. and i.v. dosing.
  • mice Male CD-1 mice were dosed with atropisomer A-2, either by i.v. administration (2 mg/kg) or by p.o. administration (10 mg/kg).
  • terminal blood samples were taken from individual animals and delivered into labelled polypropylene tubes containing anticoagulant (EDTA). The samples were held on wet ice for a maximum of 30 min while sampling of all the animals in the cohort was completed. The blood samples were centrifuged for plasma (4°C, 21100 g for 5 min) and the resulting plasma transferred into corresponding labelled tubes. Terminal brains from each PO dosed animal were excised, rinsed with saline and placed into pre-weighed labelled polypropylene tubes and the samples re-weighed prior to storage.
  • EDTA anticoagulant
  • Atropisomer A-2 shows efficacy in glioblastoma mouse models when tumours are implanted subcutaneously and orthotopically, as indicated by the studies described below.
  • U87-Luc cells were intracerebrally implanted into the brains of male athymic nude mice and tumour growth was monitored by bioluminescent signal.
  • animals were given an oral dose of 100 mg/kg of atropisomer A-2 on days 1, 4, 7, 10 and 13.
  • the control group animals were given vehicle only.
  • the results, shown in Figure 15, demonstrate a decrease in tumour signal for the treated verses the control group on Day 15.
  • Atropisomer A-2 has shown efficacy in a KRAS mutated colorectal cancer model, as described below.
  • mice bearing HCT 116 xenograft tumours were give an oral dose of 100 mg/kg atropisomer A-2 on days 1 , 8 and 15 and the tumour volumes were measured over 3 weeks. Tumour volumes in a control group of tumour- bearing mice, who had received vehicle only at the same time points were also measured.
  • a tablet composition containing a tartaric acid salt or a composition of matter according to any one of Embodiments 1.1 to 1.19 or the Examples above is prepared by mixing 50 mg of the compound with 197 mg of lactose (BP) as diluent, and 3 mg magnesium stearate as a lubricant and compressing to form a tablet in known manner.
  • BP lactose
  • a capsule formulation is prepared by mixing 100 mg of a tartaric acid salt or a composition of matter according to any one of Embodiments 1.1 to 1.19 or the Examples above with 100 mg lactose and filling the resulting mixture into standard opaque hard gelatin capsules.
  • a parenteral composition for administration by injection can be prepared by dissolving a tartaric acid salt or a composition of matter according to any one of Embodiments 1.1 to 1.19 or the Examples above in water containing 10% propylene glycol to give a concentration of active compound of 1.5 % by weight. The solution is then sterilised by filtration, filled into an ampoule and sealed.
  • a parenteral composition for injection is prepared by dissolving in water a a tartaric acid salt or a composition of matter according to any one of Embodiments 1.1 to 1.19 or the Examples above (2 mg/ml) and mannitol (50 mg/ml), sterile filtering the solution and filling into sealable 1 ml vials or ampoules.
  • a formulation for i.v. delivery by injection or infusion can be prepared by dissolving a composition of matter according to any one of Embodiments 1.1 to 1.19 or the Examples above in water at 20 mg/ml. The vial is then sealed and sterilised by autoclaving.
  • a formulation for i.v. delivery by injection or infusion can be prepared by dissolving a composition of matter according to any one of Embodiments 1.1 to 1.19 or the Examples above in water containing a buffer (e.g. 0.2 M acetate pH 4.6) at 20mg/ml. The vial is then sealed and sterilised by autoclaving.
  • a buffer e.g. 0.2 M acetate pH 4.6
  • a composition for sub-cutaneous administration is prepared by mixing a composition of matter according to any one of Embodiments 1.1 to 1.19 or the Examples above with pharmaceutical grade corn oil to give a concentration of 5 mg/ml.
  • the composition is sterilised and filled into a suitable container.
  • compositions are frozen using a one-step freezing protocol at (-45 °C).
  • the temperature is raised to -10 °C for annealing, then lowered to freezing at -45 °C, followed by primary drying at +25 °C for approximately 3400 minutes, followed by a secondary drying with increased steps if temperature to 50 °C.
  • the pressure during primary and secondary drying is set at 80 millitor.

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Abstract

La présente invention concerne un sel de (+)-L-tartrate de (R)-2,4-[5-(4-chlorophényl)-1-[2-(trifluorométhyl)-phényl]pyrrol-2-yl]-N-[2-(diméthylamino)-éthyl]benzamide, des procédés pour sa préparation, des compositions pharmaceutiques le contenant et son utilisation dans le traitement de maladies telles que le cancer.
PCT/EP2021/076203 2020-09-25 2021-09-23 Sel pharmaceutique d'un dérivé d'arylpyrrole WO2022063899A1 (fr)

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BR112023004275A BR112023004275A2 (pt) 2020-09-25 2021-09-23 Sal farmacêutico de um derivado de aril pirrol
EP21777561.8A EP4217343A1 (fr) 2020-09-25 2021-09-23 Sel pharmaceutique d'un dérivé d'arylpyrrole
KR1020237014101A KR20230074784A (ko) 2020-09-25 2021-09-23 아릴피롤 유도체의 약학적 염
IL301462A IL301462A (en) 2020-09-25 2021-09-23 A pharmaceutical salt of an arylpyrrole derivative
US18/245,999 US20240140908A1 (en) 2020-09-25 2021-09-23 A pharmaceutical salt of an arylpyrrole derivative
JP2023519234A JP2023544291A (ja) 2020-09-25 2021-09-23 アリールピロール誘導体の薬学的塩
CN202180065652.1A CN116348105A (zh) 2020-09-25 2021-09-23 芳基吡咯衍生物的药用盐
AU2021350389A AU2021350389A1 (en) 2020-09-25 2021-09-23 A pharmaceutical salt of an arylpyrrole derivative
CA3192729A CA3192729A1 (fr) 2020-09-25 2021-09-23 Sel pharmaceutique d'un derive d'arylpyrrole
MX2023003300A MX2023003300A (es) 2020-09-25 2021-09-23 Una sal farmaceutica de un derivado de arilpirrol.

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MX2023003300A (es) 2023-04-13
KR20230074784A (ko) 2023-05-31
BR112023004275A2 (pt) 2023-04-04
CN116348105A (zh) 2023-06-27
WO2022063899A9 (fr) 2022-09-22
CA3192729A1 (fr) 2022-03-31
CL2023000845A1 (es) 2023-10-20
AU2021350389A1 (en) 2023-06-01
US20240140908A1 (en) 2024-05-02

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